US12319944B2ActiveUtilityA1
Incorporation of unnatural nucleotides and methods thereof
Est. expiryJul 11, 2037(~11 yrs left)· nominal 20-yr term from priority
Inventors:Jerod PtacinCarolina E. CaffaroHans AerniYorke ZhangEmil C. FischerAaron W. FeldmanVivian T. DienFloyd E. Romesberg
C12N 15/90C12N 15/113C12Y 601/01026C12P 21/02C12N 9/93C12N 15/11
83
PatentIndex Score
0
Cited by
838
References
28
Claims
Abstract
Disclosed herein are methods, compositions and kits for the synthesis of proteins which comprises unnatural amino acids that utilize a mutant tRNA.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1. A method of producing a protein comprising an unnatural amino acid in a cell, the method comprising:
contacting the cell with an unnatural amino acid, the cell comprising:
(i) a nucleoside triphosphate transporter from Phaeodactylum tricornutum;
(ii) a tRNA from Methanosarcina mazei or Methanococcus jannaschii comprising an anticodon, the anticodon comprising an unnatural nucleotide comprising a first unnatural nucleobase independently selected from:
(iii) an aminoacyl tRNA synthetase characterized in that it aminoacylates the tRNA with the unnatural amino acid;
(iv) an mRNA encoding the protein comprising the unnatural amino acid and comprising a codon, the codon comprising an unnatural nucleotide comprising a second unnatural nucleobase independently selected from:
wherein the anticodon of the tRNA pairs with the codon of the mRNA;
wherein the mRNA is translated in the cell involving the tRNA, thereby producing a protein comprising the unnatural amino acid.
2. The method of claim 1 , further comprising introducing an unnatural nucleotide into the cell via the nucleoside triphosphate transporter.
3. The method of claim 1 , wherein the first unnatural nucleobase or the second unnatural nucleobase is
4. The method of claim 1 , wherein the second unnatural nucleobase is
and the first unnatural nucleobase is
5. The method of claim 1 , wherein the anticodon comprises the sequence GYU or GYC, wherein Y is an unnatural nucleotide comprising the first unnatural nucleobase.
6. The method of claim 5 , wherein Y is an unnatural nucleotide comprising the first unnatural nucleobase, which is
7. The method of claim 1 , wherein the aminoacyl tRNA synthetase is from Methanosarcina barkeri or Methanococcus jannaschii.
8. The method of claim 1 , wherein the cell is a microorganism or bacterium.
9. The method of claim 1 , wherein the cell is E. coli.
10. A method of producing a protein comprising an unnatural amino acid in a cell, the method comprising contacting the cell with at least one unnatural nucleotide and at least one unnatural amino acid, the cell comprising:
(i) a nucleoside triphosphate transporter from Phaeodactylum tricornutum;
(ii) a tRNA from Methanosarcina mazei or Methanococcus jannaschii comprising an anticodon, the anticodon comprising an unnatural nucleotide comprising a first unnatural nucleobase independently selected from:
(iii) an aminoacyl tRNA synthetase characterized in that it aminoacylates the tRNA with the unnatural amino acid;
(iv) a nucleic acid encoding an mRNA encoding the protein comprising the unnatural amino acid and comprising a codon, the codon comprising an unnatural nucleotide comprising a second unnatural nucleobase independently selected from:
wherein the anticodon of the tRNA pairs with the codon of the mRNA
wherein:
(a) the at least one unnatural nucleotide independently comprises the first nucleobase or the second nucleobase:
(b)(i) the mRNA comprising the codon is synthesized by transcription and the at least one unnatural nucleotide comprising the second nucleobase is incorporated into the mRNA during transcription; and/or (ii) the tRNA comprising the anticodon is synthesized by transcription and the at least one unnatural nucleotide comprising the first nucleobase is incorporated into the tRNA during transcription; and
(c) the mRNA is translated into the cell involving the tRNA, thereby producing a protein comprising the unnatural amino acid.
11. The method of claim 10 , wherein the at least one unnatural nucleotide is introduced into the cell via the nucleoside triphosphate transporter and is incorporated into the mRNA and/or the tRNA during transcription.
12. The method of claim 10 , wherein the first unnatural nucleobase or the second unnatural nucleobase of the unnatural nucleotide in the anticodon or the codon is
and/or wherein the unnatural nucleobase of the unnatural nucleotide in the mRNA is
and the unnatural nucleobase of the unnatural nucleotide in the tRNA is
and/or wherein the anticodon comprises the sequence GYU or GYC, wherein Y is an unnatural nucleotide comprising the first unnatural nucleobase.
13. The method of claim 12 , wherein Y is an unnatural nucleotide comprising the first unnatural nucleobase, which is
14. The method of claim 10 , wherein the cell is a microorganism or bacterium.
15. The method of claim 10 , wherein the cell is E. coli.
16. The method of claim 1 , wherein at least one or both of the first unnatural nucleobase and the second unnatural nucleobase are each independently selected from:
17. The method of claim 1 , wherein at least one or both of the first unnatural nucleobase and the second unnatural nucleobase are each independently selected from:
18. The method of claim 1 , wherein the codon comprises the sequence AXC or GXC, wherein X is an unnatural nucleotide comprising the second unnatural nucleobase.
19. The method of claim 18 , wherein X is an unnatural nucleotide comprising the second unnatural nucleobase, which is
20. The method of claim 1 , wherein the unnatural amino acid is p-azido-phenylalanine (pAzF) or N 6 -[(2-propynyloxy) carbonyl]-l-lysine (PrK).
21. The method of claim 10 , further comprising expressing the aminoacyl tRNA synthetase from a plasmid in the cell.
22. The method of claim 10 , wherein at least one or both of the first unnatural nucleobase and the second unnatural nucleobase are each independently selected from:
23. The method of claim 10 , wherein at least one or both of the first unnatural nucleobase and the second unnatural nucleobase are each independently selected from:
24. The method of claim 10 , wherein the codon comprises the sequence AXC or GXC, wherein X is an unnatural nucleotide comprising the second unnatural nucleobase.
25. The method of claim 24 , wherein X is an unnatural nucleotide comprising the second unnatural nucleobase, which is
26. The method of claim 10 , wherein the unnatural amino acid is p-azido-phenylalanine (pAzF) or N 6 -[(2-propynyloxy) carbonyl]-l-lysine (PrK).
27. The method of claim 10 , further comprising expressing the aminoacyl tRNA synthetase from a plasmid in the cell.
28. The method of claim 10 , wherein the aminoacyl tRNA synthetase is from Methanosarcina barkeri or Methanococcus jannaschii.Cited by (0)
No later patents cite this yet.
References (0)
No backward citations on record.